Enteropathogenic E. coli (EPEC), a cause infantile diarrhea, is associated with significant morbidity and mortality. EPEC is not invasive and does not produce toxins, but expresses a type III secretory system (TTSS) that allows translocation of bacterial proteins into host cells. EPEC disrupts intestinal epithelial tight junctions (TJ) in a TTSS-dependent manner which likely contributes to diarrhea. The overarching hypothesis of this proposal is that EPEC effector molecules work in concert to alter TJs in vitro and in vivo. The overall goal of this proposal is to define the mechanisms by which EPEC alters TJs. Several EPEC effectors appear to contribute to TJ disruption including the translocated intimin receptor (Tir) and E. coli secreted protein F (EspF). Tir is inserted into the apical host cell membrane triggering "pedestal" formation through recruitment of actin, ezrin and other cytoskeletal proteins. Tir may allow for effective translocation of effectors into host cells;alternatively, activation and recruitment of ezrin may be key. EspF binds cytokeratin 18 and 14-3-3 and is crucial for TJ disruption. EspF has also been shown to localize to mitochondria and induce apoptosis;the contribution to TJ disruption has not been examined. Our data suggest that TJ disruption can be dissected into 2 events mediated by different effectors: cytoskeletal contraction via myosin light chain (MLC) phosphorylation and endocytosis of TJ proteins. While in vitro studies have yielded important information regarding EPEC pathogenesis, corroboration of these findings in vivo has been delayed by the lack of a small animal model. We have reported the C57BL/6 mouse as an in vivo model of EPEC infection and our preliminary data suggest that intestinal TJs are disrupted. This model will allow us to determine if data from in vitro and in vivo models of EPEC infection correlate. We hypothesize that Tir-mediated intimate attachment and pedestal formation promote translocation of effectors into host cells wherein they activate signaling pathways or interact with proteins resulting in altered TJ structure and function. The following specific aims will address this hypothesis: 1. To determine the role of Tir in EPEC-induced disruption of tight junctions (TJ). 2. To investigate the relationship between EspF/14-3-3 interactions, mitochondrial targeting, and apoptosis to EPEC-induced alterations in TJ structure and barrier function. 3. To dissect the mechanisms underlying EPEC-induced disruption of TJs and define the effector molecules responsible for each component. 4. To evaluate the effect of EPEC infection on TJ structure and function in vivo using a mouse model and investigate the effector molecules and mechanisms involved.